Cellulose and hemicellulose are the most abundant plant materials produced by photosynthesis. They can be degraded and used as an energy source by numerous microorganisms (e.g., bacteria, yeast and fungi) that produce extracellular enzymes capable of hydrolysis of the polymeric substrates to monomeric sugars (Aro et al. (2001) J. Biol. Chem. 276:24309-14).
Cellulases are enzymes that hydrolyze cellulose (β-1,4-glucan or β-D-glucosidic linkages) resulting in the formation of glucose, cellobiose, cellooligosaccharides, and the like. Cellulases have been traditionally divided into three major classes: endoglucanases (EC 3.2.1.4) (“EG”), exoglucanases or cellobiohydrolases (EC 3.2.1.91; “CBH”) and β-glucosidases (β-D-glucoside glucohydrolase; EC 3.2.1.21; “BG”) (Knowles et al. (1987) TIBTECH 5:255-61; and Schulein (1988) Methods Enzymol. 160:234-43). Endoglucanases act mainly on the amorphous parts of the cellulose fibre to hydrolyze internal β-1,4-glucosidic bonds in regions of low crystallinity. Cellobiohydrolases hydrolyze cellobiose from the reducing or non-reducing end of cellulose and are able to degrade crystalline cellulose (Nevalainen and Penttila (1995) Mycota 303-319). The presence of a cellobiohydrolase (CBH) in a cellulase system is believed to be required for efficient solubilization of crystalline cellulose (Suurnakki et al. (2000) Cellulose 7:189-209). β-glucosidase acts to liberate D-glucose units from cellobiose, cellooligosaccharides, and other glucosides (Freer (1993) J. Biol. Chem. 268:9337-42). β-glucosidases have also been shown to catalyze the hydrolysis of alkyl and/or aryl beta-D-glucosides such as methy β-D-glucoside and p-nitrophenyl glucoside as well as glycosides containing only carbohydrate residues, such as cellobiose.
Cellulases are known to be produced by a large number of bacteria, yeast and fungi. Certain fungi produce complete cellulase systems that include exo-cellobiohydrolases or CBH-type cellulases, endoglucanases or EG-type cellulases and β-glucosidases or BG-type cellulases. Other fungi and bacteria express little or no CBH-type cellulases. Trichoderma reesei (also referred to as Hypocrea jecorina) expresses a large number of cellulases, including two CBHs, i.e., CBHI (Cel7a) and CBHII (Cel6a), at least eight EGs, i.e., EGI (Cel7b), EGII (Cel5a), EGIII (Cel12a), EGIV (Cel61a), EGV (Cel45a), EGVI (Cel74a), EGVII (Cel61b), and EGVIII (Cel5b), and at least five BGs, i.e., BG1 (Cel3a), BG2 (Cel1a), BG3 (Cel3b), BG4 (Cel3c) and BG5 (Cel1b). EGIV, EGVI, and EGVIII also have xyloglucanase activity.
In some cases, it is desirable to use whole cellulase broths obtained from T. reesei as a source of celluloses, e.g., for biomass conversion, textile processing, paper and pulp treatment, and the like. In other cases, T. reesei serves as an excellent host organism for the expression of engineered cellulases, cellulases from other organisms, or different enzymes or other proteins entirely (e.g., amylases, proteases, lipases, structural proteins, and the like). Particularly in these latter cases, it may be desirable to express proteins of interest in the absence of endogenous T. reesei cellulases. While this may be accomplished by deleting endogenous T. reesei cellulase genes, the large number of these genes makes such an approach time consuming and labor intensive.
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